Summary The long-term goal of this proposal is to understand the regulation of G protein-coupled receptors (GPCRs) signaling by endocytic trafficking. G protein-coupled receptors (GPCRs) comprise the largest family of signaling receptors expressed in the mammalian genome, mediate cellular responses to diverse stimuli and control vast physiological responses. Dysregulated GPCR signaling has been implicated in neurological disorders, cardiovascular diseases and cancer progression, making this receptor class the target of nearly half the drugs used clinically. In addition to desensitization, GPCR trafficking is crucial for the temporal and spatial control of receptor signaling. This is best exemplified by protease-activated receptor-1 (PAR1), a GPCR for the coagulant protease thrombin. PAR1 has important functions in vascular physiology and development as well as tumor progression and is an important drug target. Similar to most GPCRs, signaling by activated PAR1 is rapidly desensitized. We also found that activated PAR1 internalization and lysosomal sorting is critical for shutting-off PAR1 signaling. We also discovered that activated PAR1 trafficking is altered in metastatic breast carcinoma and contributes to tumor progression. However, the mechanisms responsible for PAR1 trafficking are not known. Many GPCRs are modified with ubiquitin and sorted to lysosomes through interactions with ubiquitin-binding components of the ESCRT machinery. However, not all GPCRs require ubiquitination for lysosomal sorting including PAR1. We recently discovered a novel lysosomal sorting pathway that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and is mediated ALIX. ALIX, a CHMP4/ESCRT-III interacting protein, bound to YPX3L motif of PAR1 via its V domain to facilitate lysosomal sorting. This pathway may be applicable to a subset of GPCRs containing YPXnL motifs. Our studies further indicate that the ALIX interacting protein arrestin-related protein -3 (ARRDC3) regulates PAR1 degradation. ARRDC3 appears to function as a tumor suppressor and its expression is lost in invasive breast carcinoma that exhibit dysregulated PAR1 trafficking. This proposal is focused on delineating the molecular mechanisms by which ARRDC3 and ALIX regulate GPCR ubiquitin-independent lysosomal sorting and signaling, and the contribution to breast cancer progression. The specific aims of the proposal are to: 1) determine the function of ARRDC3 in GPCR lysosomal sorting, 2) delineate the regulatory mechanisms of ALIX function in lysosomal sorting of GPCRs, and 3) examine the role of ARRDC3 in dysregulated GPCR trafficking in cancer.